Our identification and characterization of a lymphatic vascular system in the zebrafish has made it possible to bring the powerful genetic, experimental embryologic,and imaging tools available in this model organism to bear on the question of how lymphatic vessels form and what regulates their growth and assembly. Understanding how to regulate and control lymphatic vessel formation is a topic of considerable clinical interest given recent evidence suggesting that lymphatics are the major route for tumor metastasis in many if not most cancers. The long-term goal of this project is to expand on our preliminary findings in order to gain a more detailed understanding of how lymphatic vessels assemble in the fish, and then use this powerful model to study how lymphangiogenesis is regulated in vivo. The lymphatic system has become a subject of great interest in recent years because of its important role in normal and pathological processes, but progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. We demonstrated that the zebrafish possesses a lymphatic system that shares many of the morphological, molecular, and functional characteristics of the lymphatic vessels found in other vertebrates (including humans), providing a superb new model for imaging and studying lymphatic development. We also used two-photon time-lapse imaging of transgenic zebrafish to trace the origins of lymphatic progenitors, providing the first conclusive in vivo evidence that early lymphatic endothelial cells are derived from primitive venous blood vessels. We have also used the genetic and experimental tools available in the fish to show that the assembly and patterning of the larval trunk lymphatic network is orchestrated by chemokine signaling from adjacent tissues along which growing lymphatic sprouts migrate. We are currently pursuing further study of the formation of the lymphatic system through a number of ongoing projects: 1. We have generated new transgenic lines that permit direct, specific visualization of the developing lymphatic vasculature. We are using time-lapse two-photon imaging of these transgenic animals to to further characterize how the lymphatic vascular system grows and assembles during development. 2. We are carrying out forward-genetic ENU mutagenesis screens using these new lymphatic reporter transgenic lines in order to identify new mutants with defects in the development of the lympahtiv vascualr system, in order to identify novel genes that play an important role in the development of these vessels. 2. We are characterizing several mutants with lymphatic defects already identified in forward-genetic ENU mutagenesis screens. We are using exome sequencing to identify the causative genes and carrying out molecular studies to further elucidate the functino of these genes. 4. We are also carrying out reverse-genetic studies on the zebrafish functions of a number of genes previously implicated in development of the lymphatic system using mutants we have generated via TALEN- or CRISPR-mediated mutagenesis. 5. We are analyzing global patterns of lymphatic gene expression using newly developed transgenic tools facilitating lymphatic-specific gene profiling. 6. We are characterizing and studying microRNAs expressed in the lymphatic endothelium, with the goal of uncovering how these small regulatory RNAs influence gene expression in the lympahtic vasculature. The results of our studies, combining the genetic and experimental tools available in the zebrafish with the ability to perform high-resolution microscopic imaging of developing vascular structures in living animals, will continue to lead to important new insights into the origins and growth of the lymphatic system and molecular mechanisms that are critical in lymphatic development and lymphatic pathologies.